This project concerns the investigation of the detailed structure and function of an unusual heme prosthetic group present in an important class of nitrite reductase. In the previous grant period, we established the fact that this green-colored d1 heme (dioneheme) moiety has an unprecedented dioxoisobacteriochlorin (porphyrindione) structure. In the current proposal we propose to further understand the chemical, structural, spectroscopic characteristics of dioneheme, its biosynthesis, and its functions in microbial denitrification. Specifically, we propose to: 1) determine whether it is derived biosynthetically from protoporphyrin or sirohydrochlorin; 2) determine the stereochemistry, explore reactions and synthetic utilities of the quinone-like oxo groups; 3) study heme structure-enzyme function relationships in cytochrome cd1 reconstituted with synthetic dioneheme analogs, and identify specific interactions between the heme group and its protein environment; 4) study metallo- porphyrindione series compounds as models for nitrite reductase, study electrocatalytic reactions involving model hemes, and investigate reaction between dioneheme with O2 in solution and in reconstituted proteins; 5) study the electron transfer phenomenon between heme c and d1, evaluate synthetic Zn-cyt cd1 as a model biological electron transfer reactions. In order to understand the chemistry and catalytic functions of dioneheme in a broader perspective, we also plan to study the following related problems; 6) build synthetic active site models for assimilatory nitrite reductase by attaching an Fe4S4 cluster to a metalloporphyrin, with or without a bridging ligand, and characterize their structures and reactions; 7) examine porphyrin and isobacteriochlorin analogs: corphin, pyrrocorphin and porphycene, which possess either a larger or smaller core size than porphyrin; compare the physico-chemical properties of these porphyrinoids with dioneheme and siroheme; 8) characterize the vibrational modes of pi- cation radicals of porphyrin family derivatives by resonance Raman spectroscopy.